Assessment of some Herbal Drugs for Prophylaxis of Peptic Ulcer.

Aqueous (hydrophilic) and chloroform (Lipophilic) extracts of nine medicinal plants currently used in Egyptian traditional medicine to treat some gastrointestinal tract (GIT) disorders were tested for their gastro-protective effect against the incidence of peptic ulcer. Indomethacin-induced ulcer in a rat model was used for this testing. Mentha microphylla, Brassica oleracea Capitata (Cabbage), B. oleracea Botrytis (cauliflower) aqueous fraction, Portolaca oleracea polysaccharide fraction, Oreganum marjoranum, Matricaria recutita, Solanum nigrum hydrophilic and lipophilic fractions, in addition to the chloroform fraction of Portolaca oleracea and Cicorium intybus afforded high protection against the incidence of gastric ulcer (~95%). O. syriacum hydrophilic and lipophilic fractions and gum arabic afforded moderate prophylactic effect. L. sicerarea, C. intybus hydrophilic fractions and M. microphylla lipophilic fraction were inactive. Herbs represent excellent resources for cost-effective and readily available gastro-protective remedies without side effects.


Introduction
Peptic ulcer is a popular disease all over the world. It is the most common gastrointestinal tract (GIT) disorder in clinical practice. Recent survey revealed that 1.84% of population in the United States, 2.7% in Australia, and about 1.8% in Canada, Great Britain and Egypt suffer from this disease. The mortality rate, which has decreased modestly in the last few decades, is approximately 1 death per 100,000 cases (1-2). The etiology of the peptic ulcer and principles of treatments have been discussed by different authors (3)(4)(5).
The drugs currently used in the treatment of gastric ulcers are antacids, anticholinergics, proton pump inhibitors and H2-receptor antagonists (6-7). However, the majority of these drugs produce adverse reactions, such as: hypersensitivity, arrhythmia, impotence, gynecomastia and hematopoietic changes (8-9). Despite progress in conventional chemistry and pharmacology in producing effective antiulcer drugs, the plant kingdom might provide a useful source of new compounds that are used for development as pharmaceutical entities or, alternatively, as simple dietary adjuncts to existing therapies. Several herbs and spices have been used to treat GIT disorders, including gastric ulcers (3)(4)(5)(10)(11)(12)(13). The early search in the area of medicinal plant in the treatment of peptic ulcers opened the discovery of the first drug effective against peptic ulcer; carbenoxolone from Glycyrrhiza glabra (14-16), and licorice root fluid extract were used to treat followed by precipitation with ethanol. The precipitated polysaccharide fraction was washed with acetone, dried under vacuum and reserved frozen for testing. 500 g fresh B. oleracea Capitata (white cabbage) and the same weight of B. oleracea Botrytis (cauliflower) were separately homogenized with 1 L distilled water and left overnight. Solvents were, separately, evaporated under vacuum in rotary evaporator. The prepared extracts were reserved frozen for testing. Gum Arabic was used as pharmaceutical grade (El-Nasr Pharmaceutical Co., Egypt).
Another 500 g fresh B. oleracea Capitata (white cabbage) and the same weight of B. oleracea Botrytis (cauliflower) were extracted with boiling methanol (1 L, each) in a soxhlet apparatus. Air dried and powdered 200 g of M. microphylla, O. marjoranum, O. syriacum, S. nigrum, C. intybus, M. recutita, and L. cicerarea were separately extracted with hot methanol (3 x 500 mL) in a soxhlet apparatus. The solvents were, separately, evaporated under vacuum in rotary evaporator and the residue, of each plant sample, was suspended in 200 mL distilled water and partitioned with chloroform (3 x 100 mL). The combined chloroform extracts and the mother aqueous liquors were then evaporated under vacuum in rotary evaporator and residues were stored frozen for biological testing.
The crude extracts were suspended in normal saline, with the aid of tween 20 at the dose levels of 200 mg/Kg just before testing. Mucilage of P. oleracea polysccaride and gum Arabic were macerated in normal saline at the dose levels of 20 mg/Kg, 1 h before testing.

Ulcer induction with indomethacin and testing the samples
Animals, for each test, were divided into four groups of six rats each. All the animals were fasted for 24 h before the experiment. First group served as negative control and received distilled water, while second group served as positive control and received Cimetidine (250 mg/Kg). Third and fourth groups served as test groups and administered the tested sample by gastric gavage 200 mg/Kg, respectively. After 1 h of drug treatment, Indomethacin (30 mg/ Kg, p.o.) was given (21)(22). After 2 h, rats were stomach ulcers in patients had not improved with conventional medication. The glycyrrhizin of licorice was found to stop two enzymes that break down prostaglandin E (17). Effectiveness of other plant resources as cabbage in improving peptic ulcers have been reported (18)(19)(20).
The aim of the present study is the assessment of antiulcerogenic and/or gastro-protective effects of some of herbal extracts currently used in Egyptian folk medicine for some of GIT disorders.

Animals
Female Wister rats (175-200 g), were maintained on standard pellet diet and water ad libitum under standard conditions of 12 h dark-12 h light, humidity (60 ± 1.0%) and temperature (21 ± 1 °C). They were acclimatized to laboratory condition for seven days before commencement of the experiments. Fasting for 24 h was used prior to all assays because tested drugs were always administered orally by gavage (5). The experimental protocols were approved by the Institutional Animal Care and Use Committee; faculty of pharmacy, Mansoura University, Egypt.

Extract preparation
The polysaccharide fraction of fresh P. oleracea was prepared by blending 500 g of fresh herb in an electric mixer with distilled water (1 L) then lift overnight. The viscid suspension was filtered through glass wool anesthetized with ether and sacrified by cervical dislocation then the abdomen was opened, stomach excised and opened along the greater curvature, stomach contents were collected in graduated tubes to measure its volume, then stomach was washed with normal saline, and fixed in formalin. Lesions in the glandular part of the stomach were measured under an illuminated magnifying microscope (10X). Long lesions were counted and measured along their greater length in mm unit. Ptecheal lesions were counted and each five lesions were taken as 1 mm of ulcer (23). The ulcerative lesion (dark red lines) index of each animal was scored and the percentage protection was calculated according to Scarlat et al., 1985 (24). The experiment was repeated, following the same sequence, for each tested plant material.

Statistical analysis
The results were expressed as mean ± SEM. The individual data of each test and the control groups were submitted to one-way ANOVA with the level of significance set at P ˂ 0.05 (Instat-2 Computer Program: GraphPad Software Inc., V2.04, San Diego, CA, USA)

Results and Discussion
The p.o. administration of indomethacin at dose of 30 mg/Kg was sufficient to induce gastric ulcers in rats. The score of indomethacin-induced ulcer was 50.17 ± 0.47 mm and the secretions in the stomach were about 2.5 ± 0.2 mL (negative control). Oral administration of various tested plant materials before indomethacin treatment lowered the ulcer score, with some fractions, up to100% reduction (Table 1). Some plant extracts showed moderate reduction, while others were not active to protect against the gastric ulceration. Cimetidine, the known H 2 -histamine receptor antagonist was used as a positive control and showed good protection (100% reduction) against indomethacin-induced gastric ulceration (Table 1).
The results of the different experiments run for the assessment of some herbal drugs currently used for some GIT disorders are shown in Table 1 O. syriacum extracts showed moderate effect however the aqueous one was more effective than the chloroform fraction. Gum Arabic also showed moderate protection. No gastric secretions were measured for some tested fractions (Table 1) because it was very few suggesting inhibited gastric secretions. The stomachs of rats treated with chamomile aqueous fraction showed full distension with gases.
Chamomile was previously reported as protective for peptic ulcer disease (25). The aqueous extract of chamomile decreases the gastric secretions and acidity so, increases the curative ratio of gastric ulcer (26). O. marjoram is a common hot drink and P. oleracea is an edible plant whose anti-ulcerative qualities add to their house use as functional food with high medicinal values. The aqueous extract of P. oleracea was previously reported as antiulcerogenic principle (27-29). The aqueous fraction of M. microphylla showed a prominent protective effect (100% protection), which suggests the beneficial use of infusion or decoction teas of Mentha as protective against peptic ulcers. The Mentha leaves were previously reported to improve pain of nonulcer dyspepsia (30). However, Mentha extract significantly decreases the total acidity in the stomach, it doesn't affect the volume of gastric juice (31). The total alcoholic extract of O. syriacum was previously reported to have 78% protection against ethanol-induced ulcer (8). Both B. oleracea white cabbage and cauliflower fractions showed good protection against peptic ulcers, 93.6 -96.5% and 83.30 -97.3%, respectively. This confirmed the previously reported anti-ulcer effects of these plants due to increased hexosamine levels and antisecretory effect, suggesting gastric mucosal protection (18,32). While the chloroform extract of C. intybus showed 96% protection, it᾽s water soluble fraction increased the ulcer score however it has anti-Helicobacter Pylori effect (33). The titled plant roots and leaves were previously reported to have antiulcerogenic effect (34). The aerial parts of S. nigrum, powder-form and methanolic extract, were reported to decrease the ulcer index significantly due to inhibition of acid and pepsin secretions (32). The aqueous and ethanolic extracts of P. oleracea were previously reported to exhibit gastroprotective effect due to decreased gastric secretions (35).
The development of peptic ulcers can be due to imbalance between the insulting effect of acid and pepsin and the protective effect of mucosal barrier (36). Cimetidine has little cytoprotective effect but its principal action is selective antagonism of H 2 histaminic receptors and it possesses antisecretory action (37-39). It was reported to inhibit the indomethacin-induced ulcer by 94.8% (40). Other mechanisms are now hypothesized for this imbalance including the decreased gastric hexosamine level and weakness of gastric barrier (10) generation of oxygen free radical and increased peroxidation of the biological membranes (41). Indomethacin is a non-selective cyclooxygenases inhibitor known to induce gastric damage through suppression of prostaglandin generation, overproduction of leukotrienes, topical irritancy and reducing the local blood flow (42). In the stomach, prostaglandins play a vital protective role, stimulating the secretion of bicarbonate and mucus, maintaining mucosal blood flow, and regulating mucosal cell turnover and repair in addition to its action as cytoprotective (43, 44). Drugs which produce prophylaxis against the indomethacin effect can act through antagonizing all or some of its mechanisms.